The gp130/STAT3-endoplasmic reticulum stress axis regulates hepatocyte necroptosis in acute liver injury

Aim To investigate the effect of the gp130/STAT3-endoplasmic reticulum (ER) stress axis on hepatocyte necroptosis during acute liver injury. Methods ER stress and liver injury in LO2 cells were induced with thapsigargin, and in BALB/c mice with tunicamycin and carbon tetrachloride (CCl4). Glycoprotein 130 (gp130) expression, the degrees of ER stress, and hepatocyte necroptosis were assessed. Results ER stress significantly upregulated gp130 expression in LO2 cells and mouse livers. The silencing of activating transcription factor 6 (ATF6), but not of ATF4, increased hepatocyte necroptosis and mitigated gp130 expression in LO2 cells and mice. Gp130 silencing reduced the phosphorylation of CCl4-induced signal transducer and activator of transcription 3 (STAT3), and aggravated ER stress, necroptosis, and liver injury in mice. Conclusion ATF6/gp130/STAT3 signaling attenuates necroptosis in hepatocytes through the negative regulation of ER stress during liver injury. Hepatocyte ATF6/gp130/STAT3 signaling may be used as a therapeutic target in acute liver injury.

The liver is prone to injury by pathogens and toxins (1). Liver injury can induce compensative anti-injury responses, such as endoplasmic reticulum (ER) stress in hepatocytes, to reduce their degeneration and necrosis (2). These defense responses can mitigate the sensitivity of hepatocytes to injury stimuli (3). However, the mechanisms through which they modulate ER stress-related liver injury have not been clarified.
Necroptosis is a mode of programmed cell death, characterized by a rupture of cell membranes and release of cell contents (4). Although necroptosis shows morphological changes similar to necrosis, it is regulated by a specific molecular mechanism (5). Biochemical markers of necroptosis are the RIPK1-RIPK3 complex formation and mixed lineage kinase domain-like pseudokinase (MLKL) phosphorylation. Furthermore, the release of cell contents leads to pro-inflammatory cell death and hepatocyte injury (6). Therefore, effective control of necroptosis can reduce liver injury. ER stress, activated by various physiological and pathological conditions (7), contributes to the pathogenesis of liver diseases (8,9). During ER stress, unfolded proteins accumulate in the ER lumen and bind to glucose-regulated protein 78 (GRP78), which promotes the activation of protein kinase R-like ER kinase (PERK) and inositol requiring enzyme 1 (IRE1), and the translocation of activating transcription factor 6 (ATF6) (10). ATF6 acts as a transcription factor and induces the expression of ER chaperone proteins (11). Overall, ER stress represses protein synthesis by activating PERK/eIF2α signaling (12), IRE1 endonuclease activity (13), and ER-associated degradation (ERAD) (14), as well as promotes cell survival. However, persistent or aberrant ER stress can induce cell injury, such as caspase-dependent cell apoptosis and caspase-independent necroptosis. ER stress mediates hepatocyte apoptosis by activating CHOP and caspase-12, so that the protein levels of CHOP and caspase 12 reflect the levels of ER stress.
In this study, we investigated the effect of ER stress on gp130 expression in mouse models of acute liver injury induced by tunicamycin (TM, an ER stress inducer) and carbon tetrachloride (CCl 4 ), and in a cellular model of ER stress induced by thapsigargin (TG). In addition, we investigated the impact of GP130 silencing on ER stress, necroptosis, and liver injury. To test ER stress and gp130 expression, the untreated control mice were injected intraperitoneally with phosphate buffer saline (PBS, 10 mL/kg, TM solvent) or olive oil (5 mL/ kg, CCl 4 solvent); the TM group with 2 mg/kg TM (Sigma, St. Louis, MO, USA) for 24 h or 48 h; and the CCl 4 group with a 5 mL/kg-mixture containing 1 mL CCl 4 (Sigma) and 4 mL olive oil, for 24 h or 48 h. TM is an ER stress inducer that hinders glycosylation of nascent proteins in the ER (28,29). CCl 4 can induce liver injury through its direct effect and its metabolic products of free radicals. Particularly, CCl 3 , which is produced by the decomposition of CCl 4 , can cause lipid peroxidation, leading to cell membrane damage (30). Accordingly, the groups were as follows: untreated, 24 h TM and its solvent control, and 48 h TM and its solvent control (5 groups, total = 60 mice); untreated, 24 h CCl 4 and its solvent control, and 48 h CCl 4 and its solvent control (5 groups, total = 60 mice). The protocol was based on our preliminary study.

Histochemical and immunohistochemical analysis
The paraffin-embedded liver tissue sections (5 μm) were routinely stained with hematoxylin and eosin (32) and scanned with a panoramic slice scanner (Pannoramic DESK/MIDI/250/1000, 3DHISTECH, Budapest, Hungary). The generated images were viewed with CaseViewer2.3 software (3DHISTECH). After background adjustment, the necrotic tissue areas were measured with Image-Pro Plus 6.0 software in three visual fields (33). The liver tissue sections were scored by two experienced pathologists using the Histology Activity Index-Knodell score in a blinded manner (34,35). Additionally, the liver tissue sections were stained with anti-gp130 antibody (sc-376280, 1:100; Santa Cruz Biotechnology) and visualized with the diaminobenzidine (DAB) reagent (PV-9002, ZSBIO, Beijing, China).

serum alanine aminotransferase and total bilirubin levels
The levels of serum alanine aminotransferase (ALT) and total bilirubin (TBil) were measured with a Beckman Coulter auto-analyzer (AU5800, Beckman Coulter, Brea, CA, USA) (36).

analysis of aTF6 binding to the GP130 promoter
The promoter sequence of human GP130 gene at its transcription start site of 2000 bp was retrieved from the UCSC website (http://genome.ucsc.edu/index.html). Bioinformat-  ic analysis (https://jaspar.genereg.net/) was used to identify the binding sites of ATF6 in the GP130 promoter with a relative score of >0.75. The relative score of each binding site was based on the corresponding algorithm calculation. A higher score implied a greater likelihood of ATF6 binding (37).

statistical analysis
The normality of distribution was assessed with a one-sample Kolmogorov-Smirnov test. Data are presented as mean ± standard deviation (SD). The differences among the groups were assessed with a least significant difference (LSD), one-way analysis of variance (ANOVA) and a post-hoc LDS. A P-value of <0.05 was considered statistically significant (38). The analysis was performed with SPSS 18.0 (SPSS, Chicago, IL, USA).  dent manner (P = 0.036, and P < 0.001 at 24 and 48 h, respectively; Figure 1A). It also increased MLKL and eIF2α phosphorylation, as well as ATF4, ATF6, and gp130 protein expression (P < 0.001; Figure 1B and 1C, respectively). Hence, ER stress upregulated gp130 expression and necroptosis in hepatocytes.
The silencing of aTF6, but not of aTF4, reduces gp130 expression in Lo2 cells after inducing er stress ATF4 or ATF6 silencing significantly decreased the viability of LO2 cells (P = 0.017 and P = 0.028, respectively) and further decreased the viability of TG-treated LO2 cells (P = 0.006 and P = 0.009, respectively; Figure 2A and B). Fur- thermore, it decreased ATF6 or ATF4 expression by 15%-60%. ATF6, but not ATF4, silencing significantly mitigated gp130 expression upregulated by TG ( Figure 2C and D). This finding suggests that ER stress upregulates gp130 expression, partially depending on ATF6 expression. More-over, ATF6 silencing significantly increased the relative levels of MLKL phosphorylation in both control and TGtreated LO2 cells (P < 0.001; Figure 2E), a finding that suggests that ATF6 attenuates hepatocyte necroptosis. Bioinformatic analysis using JASPAR predicted two binding sites in the human GP130 promoter for human ATF6, based on the relative profile score of ≥75% (Table 4). In conclusion, gp130 expression upregulated by ER stress may depend on ATF6 expression in LO2 cells.

TM and CCl 4 induce er stress and upregulate gp130 expression in mouse hepatocytes
Compared with the vehicle-injected and untreated controls, mice injected with TM had significantly elevated levels of serum ALT and TBil, increased necrotic areas in the liver, and enhanced MLKL phosphorylation (P < 0.001 for all; Figure 3A-D). This indicates that TM injection induces acute liver injury, likely resulting in hepatocyte necroptosis. Interestingly, TM injection also significantly enhanced eIF2α phosphorylation, as well as ATF4, ATF6, and gp130 protein expression (P < 0.001 for all; Figure 3E). Immunohistochemistry further demonstrated that TM injection significantly upregulated gp130 expression (P < 0.001; Figure  3F). Similarly, CCl 4 injection induced liver injury, hepatocyte necroptosis, and upregulated p-MLKL, p-eIF2α, ATF4, ATF6, and gp130 protein expression (P < 0.001; Figure 3G-L). These findings demonstrate that ER stress not only induces acute liver injury but also upregulates gp130 expression in the mouse liver.
atf6 silencing mitigates gp130 expression in the liver tissues of CCl 4 -injected mice First, Atf6 silencing alone did not significantly change the levels of serum ALT and TBil, but did further significantly increase the levels of serum ALT and TBil in the mice injected with CCl 4 (P = 0.026 and P < 0.001, respectively; Figure 4A and B). This suggests that Atf6 silencing deteriorates CCl 4 -damaged liver function. In parallel, Atf6 silencing significantly increased the necrotic areas (P < 0.001; Figure 4C) and further elevated MLKL phosphorylation (P < 0.001 for both; Figure  4D). More importantly, Atf6 silencing not only significantly decreased ATF6 levels and gp130 expression, but also mitigated CCl 4 -upregulated gp130 expression (P < 0.001; Figure  4E). In conclusion, Atf6 silencing deteriorates acute liver injury induced by ER stress and mitigates CCl 4 -upregulated gp130 expression. The gp130 upregulated expression by CCl 4 partially depends on ATF6 expression.

Gp130 silencing aggravates CCl 4 -induced hepatocyte er stress and necroptosis in mice
Gp130 silencing did not significantly alter the levels of serum ALT and TBil in control mice, but it did significantly increase serum ALT and TBil levels in CCl 4injected mice (P = 0.039 and P = 0.043, respectively; Figure 5A and B). Furthermore, Gp130 silencing significantly increased the necrotic areas in the livers of CCl 4 -injected mice (P = 0.036; Figure 5C) and enhanced MLKL phosphorylation in the livers of both control and CCl 4 -injected mice (P < 0.001 for both; Figure 5D). These findings indicate that gp130 may protect from CCl 4 -induced hepatocyte necrosis, particularly from necroptosis in mice. In addition, Gp130 silencing significantly decreased gp130 levels and STAT3 expression, as well as STAT3 phosphorylation, in both control and CCl 4 -injected mice (P < 0.001 for all; Figure 5E). Finally, it dramatically increased CHOP levels and caspase-12 expression in CCl 4 -injected mice (P < 0.001, for both; Figure 5F). Together, these data indicate that Gp130 silencing attenuates gp130/STAT3 signaling, and deteriorates CCl 4 -induced ER stress, hepatocyte necroptosis, and acute liver injury in mice.

DisCussion
In this study, ER stress upregulated gp130 protein expression in hepatocytes during acute liver injury. Furthermore, ATF6 silencing aggravated ER stress-mediated hepatocyte necroptosis, and mitigated gp130 expression both in vitro and in vivo. Apparently, ER stress enhanced gp130 expression in hepatocytes, partially depending on ATF6. Interestingly, Gp130 silencing also mitigated CCl 4induced STAT3 phosphorylation, as well as aggravated liver injury, hepatocyte necroptosis, and ER stress. This suggests that increased gp130 expression may protect hepatocytes from necroptosis by enhancing STAT3 activation. Therefore, hepatocyte ATF6/gp130/STAT3 signaling mitigates necroptosis during acute liver injury and may relieve ER stress.
Gp130 expression is upregulated in various types of tumors and non-neoplastic liver diseases (39,40). It can be regulated by multiple factors (41). First, gp130 binds cytokines and other molecules to transmit signals, a process leading to the endocytosis of gp130 and its ligand to avoid its sustained activation in a specific sequence-dependent manner (42,43). In addition, the stability of gp130 activity is regulated by its phosphorylation, ubiquitination, and various post-transcriptional modifications as gp130 can be degraded in the lysosomal or proteasome pathway (44,45). Moreover, ER stress can downregulate protein synthesis by enhancing PERK/eIF2α/ATF4 signaling, IRE1 endonuclease activity, and ERAD (46). Given that gp130 is a transmembrane protein, its synthesis requires the ER (47). Hence, ER stress may inhibit gp130 expression. However, our results indicated that ER stress upregulated gp130 expression in human hepatocytes and mouse livers. The differences in these findings may stem from different experimental conditions.
ER stress is mediated by PERK/eIF2α/ATF4, ATF6, and IRE1 signaling (48). PERK phosphorylates the serine Ser51 site of eIF2α, which acts as a competitive inhibitor of eIF2B and consequently inhibits the overall protein translation in the cell, thereby reducing the ER burden (49). At the same time, eIF2α can selectively initiate the expression of ATF4, which is a transcription factor, and can further induce GRP78 and CHOP expression to positively regulate ER stress (50,51). However, in our study, eIF2α phosphorylation, triggered by ER stress during acute liver injury, did not inhibit gp130 expression, and ATF4 silencing did not significantly alter TG-upregulated gp130 expression in LO2 cells. This indicates that gp130 expression upregulated by ER stress is independent of ATF4 activity in hepatocytes.
ER stress can induce ATF6 in the Golgi apparatus to enter the nucleus, thereby promoting the expression of ER-related degradation proteins, which degrade unfolding proteins by binding to the ER stress response elements (ERSE-I, ERSE-II), UPR elements, and cAMP response elements in the promoters (52,53). ATF6 activity determines the cell fate decision between survival and death (54). In this study, ATF6 silencing mitigated gp130 expression upregulated by ER stress in human hepatocyte cell line and mouse livers. Furthermore, bioinformatic analysis using JASPAR found two binding sites of ATF6 with a relative score of >0.75 within the GP130 promoter. These findings suggest that ATF6 may directly enhance GP130 transcription during ER stress. Gp130/STAT3 signaling is crucial for the pathogenesis of several liver diseases (55). Hepatocyte-specific gp130 knockout mice are prone to acute liver injury (56). Moreover, inhibition of IL-6/gp130 signaling can benefit patients with liver cancer (57,58). This implies that elevated gp130 expression may mitigate liver injury. In contrast, IL-6/gp130 signaling may aggravate liver damage in autoimmune hepatitis (59).  (70). However, it also upregulated RIP3 expression, which did not alleviate necroptosis of liver cells (69). Therefore, although ATF6 alleviates hepatocyte necroptosis during liver injury, its specific regulatory effect is extremely complex and remains to be investigated.
This study discovered that Gp130/STAT3 signaling in hepatocytes has a protective effect on the liver. However, in clinical practice, IL-6 treatment for liver damage did not achieve the expected effect. Conversely, inhibiting the activity of Gp130/STAT3 signaling has been successfully applied in the treatment of inflammatory diseases in clinical practice. This suggests that the contribution of Gp130/ STAT3 signaling to the progression of liver damage in different cells may be inconsistent or even damaging. For example, the activation of Gp130/STAT3 signaling in Kupffer cells may enhance their pro-inflammatory responses and aggravate liver damage. Therefore, further research on the specific roles of the Gp130/STAT3 signaling in different cells during liver damage is needed. Also, interventions to enhance Gp130/STAT3 activity in hepatocytes while reducing it activity in pro-inflammatory cells (such as Kupffer cells) or fibrosis-promoting cells (such as hepatic stellate cells) may have more significant clinical translational implications. Additionally, the regulation of Gp130 by ATF6 should be confirmed by dual fluorescent reporter gene detection results and more in vitro experiments, such as primary liver cell experiments.
In conclusion, our data indicate that gp130/STAT3 signaling modulated ER stress during liver injury, which reduced hepatocyte necroptosis. ER stress upregulated gp130 expression by activating ATF6 in hepatocytes. The upregulated gp130 expression increased STAT3 activation and mitigated hepatocyte necroptosis and liver injury, which may be related to a negative feedback alleviating ER stress (Figure 6). Therefore, our findings may provide new insights into anti-liver injury responses during ER stress-induced acute liver injury.
Funding This study was partially supported by the grants from the Science and Technology Research Foundation of Guizhou Province or Zunyi City (QKHJC-ZK(2022)YB642, QKH · PTRC(2017)5733-013, gzwjkj2020-1-041, ZSKH · HZ(2022)344, and ZSKH · HZ(2022)360). The funders had no role in the design of the study; collection, analyses, or interpretation of data; manuscript writing; or the decision to publish the results. Declaration of authorship XL, JW conceived and designed the study; XL, JW, YL, WH acquired the data; all authors analyzed and interpreted the data; all authors drafted the manuscript; QJC, XLiu critically revised the manuscript for important intellectual content; all authors gave approval of the version to be submitted; all authors agree to be accountable for all aspects of the work.
Competing interests All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.